Developing and validating a new tool for simultaneous multi-channel wide-field imaging

开发并验证同步多通道宽视场成像的新工具

• 批准号: BB/M019144/1
• 负责人: Neil Kad
• 金额: $ 19.01万
• 依托单位: University of Kent
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2015
• 资助国家: 英国
• 起止时间: 2015 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FM019144%2F1
• 关键词: Developing; validating; new; tool; simultaneous

Imaging biological systems provides a direct insight into how they work. As more systems are being studied there is an increasing necessity to label and visualise more components. However, despite there now being a large variety of colours with which to label proteins our ability to simultaneously visualise them is limited. There are two major methods to image in multiple colours, wide-field and confocal. The former approach involves taking a picture of the whole sample at once, but is limited to only four colours per image. The latter approach involves taking pictures in one location before moving to another position to take a second image and so on until the whole sample is catalogued. Clearly this confocal approach means that the whole sample cannot be imaged simultaneously, however it does permit many colours to be imaged. In this proposal we are developing a new method to image six-colours simultaneously in wide-field, with the hope that this could be further extended in the future to many more colours. To achieve this, we will build a simple device that attaches onto a standard microscope, this will allow two commercially available 3-colour splitters to be attached and then two cameras. These cameras need to talk to each other so that they take images at the same time, we will build this interface using software or hardware. The six-colour imager developed here will then be tested using a biological system. In our lab we study excerpts from the bacterial nucleotide excision DNA repair (NER) process using a three colour imaging device. A six-colour imager will enable us to study simultaneously all of the NER proteins that act together to repair DNA damage. Our approach is to use DNA tightropes, which are nanowires of DNA suspended between microscopic platforms on a microscope coverslip. To these tightropes we will add all of the proteins involved in NER but each type will be labelled with a different coloured tag. We will then study how DNA is repaired one molecular complex at a time. As a test for the new technology developed here this model system is ideal because it requires multiple colour imaging and the highest level of detection sensitivity. We anticipate that this work will pave the way for the study of many different biological systems with multiple components, not only at the single molecule level but also at the cellular level as a consequence of wide-field imaging. This is a hugely important tool that needs to be developed and tested.

对生物系统进行成像可以直接了解它们是如何工作的。随着越来越多的系统被研究，越来越有必要标记和可视化更多的组件。然而，尽管现在有很多种颜色来标记蛋白质，但我们同时可视化它们的能力有限。有两种主要的方法来成像在多个颜色，宽场和共焦。前一种方法涉及一次拍摄整个样品的照片，但每个图像仅限于四种颜色。后一种方法涉及在一个位置拍照，然后移动到另一个位置拍摄第二张图像，依此类推，直到整个样本被编目。显然，这种共焦方法意味着整个样品不能同时成像，但它确实允许许多颜色成像。在这个提议中，我们正在开发一种新的方法，在宽视场中同时成像六种颜色，希望将来可以进一步扩展到更多的颜色。为了实现这一点，我们将建立一个简单的设备，连接到一个标准的显微镜，这将允许两个商业上可用的3色分裂器连接，然后两个相机。这些摄像头需要相互通信，以便同时拍摄图像，我们将使用软件或硬件构建此接口。这里开发的六色成像仪将使用生物系统进行测试。在我们的实验室中，我们使用三色成像设备研究了细菌核苷酸切除DNA修复（NER）过程的摘录。六色成像仪将使我们能够同时研究所有一起修复DNA损伤的NER蛋白。我们的方法是使用DNA tightropes，它是悬浮在显微镜盖玻片上的显微镜平台之间的DNA纳米线。在这些tightropes中，我们将添加NER中涉及的所有蛋白质，但每种类型将用不同颜色的标签标记。然后我们将研究DNA是如何一次修复一个分子复合物的。作为对新技术的测试，该模型系统是理想的，因为它需要多种颜色成像和最高水平的检测灵敏度。我们预计，这项工作将铺平道路，为许多不同的生物系统的研究与多个组件，不仅在单分子水平，而且在细胞水平的结果，宽场成像。这是一个非常重要的工具，需要开发和测试。

项目成果

The TFIIH subunits p44/p62 act as a damage sensor during nucleotide excision repair.

• DOI: 10.1093/nar/gkaa973
• 发表时间: 2020-12-16
• 期刊: Nucleic acids research
• 影响因子: 14.9
• 作者: Barnett JT;Kuper J;Koelmel W;Kisker C;Kad NM
• 通讯作者: Kad NM

Single-molecule imaging reveals how mavacamten and PKA modulate ATP turnover in skeletal muscle myofibrils.

• DOI: 10.1085/jgp.202213087
• 发表时间: 2023-01-02
• 期刊: The Journal of general physiology

An Approach to Derive Functional Peptide Inhibitors of Transcription Factor Activity.

• DOI: 10.1021/jacsau.2c00105
• 发表时间: 2022-04-25
• 期刊: JACS AU
• 影响因子: 8
• 作者: Brennan, Andrew;Leech, James T;Kad, Neil M;Mason, Jody M
• 通讯作者: Mason, Jody M

Understanding the coupling between DNA damage detection and UvrA's ATPase using bulk and single molecule kinetics.

• DOI: 10.1096/fj.201800899r
• 发表时间: 2019-01
• 期刊: FASEB journal : official publication of the Federation of American Societies for Experimental Biology
• 作者: Barnett JT;Kad NM
• 通讯作者: Kad NM

Single-Molecule Methods for Nucleotide Excision Repair: Building a System to Watch Repair in Real Time.

• DOI: 10.1016/bs.mie.2017.03.027
• 发表时间: 2017
• 期刊: Methods in enzymology
• 作者: Kong M;Beckwitt EC;Springall L;Kad NM;Van Houten B
• 通讯作者: Van Houten B